Gene delivery using recombinant adenoviral vectors is over a decade old (Berkner, Biotechniques 6:616-628 (1988)). There are three primary methods of generating such vectors. First, overlap recombination can be effected in the 293 helper cell line between a restriction digested adenovirus and a plasmid containing adenoviral sequences (see, for example, Hearing and Shenk, Cell 33:695-703 (1983)). Alternatively, overlap recombination can be effected in 293 cells between a 40 kb plasmid that contains the majority of the adenoviral genome and a smaller plasmid containing certain adenoviral sequences (Ghosh-Chaudhury et al, Gene 50:161-171 (1986)). Finally, a segment of DNA containing adenoviral sequences can be ligated with a restriction-digested adenovirus (Stratford Perricaudet et al, J. Clin. Invest. 90:626-630 (1992)).
In the foregoing methods, the adenoviral sequences referred to are the viral terminal repeat, which is the adenoviral DNA polymerase binding site necessary for replication, and the E1a enhancer/packaging signal that is necessary for viral assembly. The first two of the foregoing methods require additional adenoviral sequences for overlap recombination to occur in the 293 cell line.
There are problems inherent in each of the existing methods. First, none of the methods described above has a screening capacity built into it for determining the success of a particular recombination or ligation event. Further, the first and last of the methods described above use wild type virus to generate the vector backbone. The wild type virus is hazardous to use as it is replication efficient which gives it a distinct growth advantage over recombinant virions, especially large recombinant virions (Graham and Prevec, Gene Transfer and Expression Protocols, Methods in Molecular Biology 7:109-128, Humana Press (1991)). This latter problem manifests itself when an effort is made to generate recombinant virions and the restriction digestion of the viral DNA is less than complete. Even at a 99.99% complete digestion of 1 .mu.g of viral DNA, approximately 4.times.10.sup.6 copies of uncut wild-type viral DNA is present that could infect cells. This problem would hopelessly confound the isolation of recombinant virions.
The present invention provides an adenoviral vector system that is free of the problems of the systems of the art. The present system simplifies the cloning of genes into plasmid vectors, it makes possible the use of either ligation or overlap recombination in the generation of a recombinant virus and it eliminates the use of cell replication efficient viral forms.